Dynamics and orientation of N ⁺ (CD ₃ ) ₃ -bromoacetylcholine bound to its binding site on the nicotinic acetylcholine receptor

Abstract

Dynamic and structural information has been obtained for an analogue of acetylcholine while bound to the agonist binding site on the nicotinic acetylcholine receptor (nAcChoR), using wide-line deuterium solid-state NMR. Analysis of the deuterium lineshape obtained at various temperatures from unoriented nAcChoR membranes labeled with deuterated bromoacetylcholine (BAC) showed that the quaternary ammonium group of the ligand is well constrained within the agonist binding site when compared with the dynamics observed in the crystalline solids. This motional restriction would suggest that a high degree of complementarity exists between the quaternary ammonium group of the ligand and the protein within the agonist binding site. nAcChoR membranes were uniaxially oriented by isopotential centrifugation as determined by phosphorous NMR of the membrane phospholipids. Analysis of the deuterium NMR lineshape of these oriented membranes enriched with the nAcChoR labeled with N ؉ (CD3)3-BAC has enabled us to determine that the angle formed between the quaternary ammonium group of the BAC and the membrane normal is 42°in the desensitized form of the receptor. This measurement allows us to orient in part the bound ligand within the proposed receptor binding site.2 H NMR ͉ structure ͉ 31 P NMR T he ligand gated ion channel, the nicotinic acetylcholine receptor (nAcChoR), is a member of the four-transmembrane superfamily of receptors that includes ␥-aminobutyric acid, glycine, and the 5-hydroxytryptamine receptors and is involved in the transmission of information across the neuromuscular junction (1). Structurally the nAcChoR is composed of five glycosylated subunits (␣ 2, ␤,␥, ␦) with a total molecular mass of 280 kDa (1). Electron diffraction studies have been used to resolve the structure of the receptor to 4.6 Å and to define how it is conformationally altered upon the binding of acetylcholine (ACh) to the synaptic surface of the protein (2). However, the resolution of the current structures is insufficient to resolve the bound agonist, hindering a molecular understanding of the structural and dynamic events associated with the ACh binding to the agonist site on the nAcChoR.Solid-state NMR has demonstrated its applicability to the study of various dynamic processes occurring within the protein backbone, side chains, and bound ligands (3-11). Furthermore, deuterium NMR has found widespread application to the study of protein dynamics both in soluble proteins in the solid state and membrane proteins in their native environment (3-11). In this study, we have exploited the selective reactivity of ␣-Cys-192͞193 toward bromoacetylcholine (BAC) (12-14) after the reduction of the nAcChoR to covalently incorporate N ϩ (CD 3 ) 3 -BAC. The incorporation of this agonist analogue containing NMR-sensitive nuclei into the agonist binding site has allowed a detailed study of the structure and dynamics of the ligand within the agonist binding site of the nAcChoR. Using the well-characterized lineshapes of deuterated quaternary ammonium com...

The conformation of acetylcholine at its target site in the membrane-embedded nicotinic acetylcholine receptor

“…moacetylcholine within the binding site (16) and suggest that ACh binding results in conformational changes that hinder the free rotation of the quaternary ammonium group as observed in earlier 2 H spectra (16), with little hindrance from interactions with the binding site.…”

“…This analysis gives the distance between the spins at sites 1 or 2 and the three spins of site 5 at a ''pseudoatom'' located at the geometric center between the three methyl groups attached to the quaternary ammonium. At the temperatures at which these experiments were performed earlier, 2 H NMR studies (16) indicate the quaternary ammonium group is free to rotate in the binding site, suggesting that on the time scale of the experiments, an averaged dipolar coupling would be observed. We assume that the distances from sites 1 or 2 to this pseudoatom are related by the normal dipolar coupling constant with an r Ϫ3 dependence.…”

Loop 3 of Short Neurotoxin II is an Additional Interaction Site with Membrane-bound Nicotinic Acetylcholine Receptor as Detected by Solid-state NMR Spectroscopy

“…These differences may account for the low affinity of the short forms to neuronal nAChR and AChBP. [1,[3][4][5][6][7][8][9][10][11][12][13] C]glycerol as sole nitrogen and carbon sources, respectively. Gene expression was induced by increasing temperature to 37°C.…”

“…6,8,10 Solution and solid-state NMR spectroscopy have been used to characterize the binding properties of nAChR ligands. [11][12][13] From this wealth of data, the interaction site within the receptor was postulated to be located mainly on the α-subunits at the extracellular part of the muscle-type nAChR, and overlaps to a great extent with the binding pocket of acetylcholine, the natural agonist of nAChRs. 2,10 The data were used by Mordvintsev et al to construct a computational model for the complex of NTII and the extracellular domain of the muscle-type nAChR.…”

Solid‐state NMR for the analysis of high‐affinity ligand/receptor interactions

“…the exposure of this protein to light and the roles this may play in the function of this protein as a proton pump (150). Subsequently, similar techniques have been employed to analyze a range of systems including the retinal chromophore in bovine rhodopsin (151) and the agonist analog bromoacetylcholine within the agonist binding site of the nicotinic acetylcholine receptor (152).…”